Mucolipin-3 (MCOLN3) is a cation channel that belongs to the superfamily of transient receptor potential (TRP) channels. A gain-of-function mutation in MCOLN3 results in the varitint-waddler (Va) phenotype in mice, which is characterized by hearing loss, vestibular dysfunction (circling behavior, head-bobbing, waddling), and coat color dilution. Whole-cell patch-clamp techniques in cells heterologously expressing MCOLN3 revealed that is an inwardly rectifying Ca2+-permeable cation channel which activity is inhibited by acidic extracellular (or luminal) pH and increased by incubation of cells in low Na+ medium. The Va phenotype is caused by a point mutation (V419P) in the pore region that locks the channel in an open conformation. It has been suggested that the Va mutant causes massive entry of Ca2+ inside cells leading to apoptosis and cell death. Endogenous MCOLN3 mainly localizes to intracellular vesicles in hair cells, while lower levels of the protein are also observed at the plasma membrane of the stereocilia. In HeLa and human fibroblasts, endogenous MCOLN3 was found to be distributed along the endocytic pathway. In agreement with these studies, heterologously expressed MCOLN3 co-localizes with early and late endosomes/lysosomes markers in HeLa and ARPE-19 cells. Over-expression of MCOLN3 causes severe alterations in the endosomal pathway, including enlargement and clustering of endosomes, delayed Epidermal Growth Factor Receptor (EGFR) degradation, and impaired autophagosome maturation, thus suggesting that MCOLN3 plays an important role in the regulation of endosomal function. To better understand the physiological role of MCOLN3 we inhibited MCOLN3 function by expression of a channel-dead dominant negative mutant (458DD/KK) or by knockdown of endogenous MCOLN3. We found that impairment of MCOLN3 activity caused a significant accumulation of luminal Ca2+ at endosomes. This accumulation led to severe defects in endosomal acidification as well as to increased endosomal fusion. Our findings reveal a prominent role for MCOLN3 in regulating Ca2+ homeostasis at the endosomal pathway and confirm the importance of luminal Ca2+ for proper acidification and membrane trafficking.